Nanobubbles are Non-Echogenic for Fundamental-Mode Contrast-Enhanced Ultrasound Imaging.

Microbubbles (1-10 µm diameter) have been used as conventional ultrasound contrast agents (UCAs) for applications in contrast-enhanced ultrasound (CEUS) imaging. Nanobubbles (<1 µm diameter) have recently been proposed as potential extravascular UCAs that can extravasate from the leaky vasculature of tumors or sites of inflammation. However, the echogenicity of nanobubbles for CEUS remains controversial owing to prior studies that have shown very low ultrasound backscatter. We hypothesize that microbubble contamination in nanobubble formulations may explain the discrepancy. To test our hypothesis, we examined the size distributions of lipid-coated nanobubble and microbubble suspensions using multiple sizing techniques, examined their echogenicity in an agar phantom with fundamental-mode CEUS at 7 MHz and 330 kPa peak negative pressure, and interpreted our results with simulations of the modified Rayleigh-Plesset model. We found that nanobubble formulations contained a small contamination of microbubbles. Once the contribution from these microbubbles is removed from the acoustic backscatter, the acoustic contrast of the nanobubbles was shown to be near noise levels. This result indicates that nanobubbles have limited utility as UCAs for CEUS.

Growth hormone-releasing hormone is produced by adipocytes and regulates lipolysis through growth hormone receptor.

BACKGROUND: Growth hormone-releasing hormone (GHRH) has a crucial role in growth hormone (GH) secretion, but little is known about its production by adipocytes and its involvement in adipocyte metabolism. OBJECTIVES: To determine whether GHRH and its receptor (GHRH-R) are present in human adipocytes and to study their levels in obesity. Also, to analyze the effects of GHRH on human adipocyte differentiation and lipolysis. METHODS: GHRH/GHRH-R and GH/GH-R mRNA expression levels were analyzed in human mature adipocytes from non-obese and morbidly obese subjects. Human mesenchymal stem cells (HMSC) were differentiated to adipocytes with GHRH (10-14-10-8 M). Adipocyte differentiation, lipolysis and gene expression were measured and the effect of GH-R silencing was determined. RESULTS: Mature adipocytes from morbidly obese subjects showed a higher expression of GHRH and GH-R, and a lower expression of GHRH-R and GH than non-obese subjects (P<0.05). A total of 10-14-10-10 M GHRH induced an inhibition of lipid accumulation and PPAR-γ expression (P<0.05), and an increase in glycerol release and HSL expression (P<0.05) in human differentiated adipocytes. A total of 10-12-10-8 M GHRH decreased GHRH-R expression in human differentiated adipocytes (P<0.05). A total of 10-10-10-8 M GHRH increased GH and GH-R expression in human differentiated adipocytes (P<0.05). The effects of GHRH at 10-10 M on adipocyte differentiation and lipolysis were blocked when GH-R expression was silenced. CONCLUSIONS: GHRH and GHRH-R are expressed in human adipocytes and are negatively associated. GHRH at low doses may exert an anti-obesity effect by inhibiting HMSC differentiation in adipocytes and by increasing adipocyte lipolysis in an autocrine or paracrine pathway. These effects are mediated by GH and GH-R.

Rufinamide in children and adults in routine clinical practice.

OBJECTIVE: To explore the long-term effectiveness of rufinamide in managing Lennox-Gastaut Syndrome (LGS), other epileptic encephalopathies, and intractable focal epilepsies in adults and children in routine clinical practice. METHODS: A multicentre, retrospective chart review of patients prescribed adjunctive rufinamide at seven Spanish epilepsy centres, with assessments at six and 12 months. RESULTS: We evaluated data from 58 patients (40 male, age range 7-57 years), 25 of whom were diagnosed with LGS, 12 with other epileptic encephalopathies and 21 of whom were diagnosed with focal epilepsies, mainly frontal lobe. The mean daily rufinamide dose was 32.0 mg/kg (range 12.5-66.7 mg/kg) in children and 24.7 mg/kg (range 5.0-47.0 mg/kg) in adults, and the most commonly used concomitant antiepileptic drugs were levetiracetam and valproate. Rufinamide was discontinued in 25 patients (43.1%) during the 1-year follow-up, and the most common reason was lack of effectiveness (n = 12, 20.7% of total). The frequency of generalized tonic-clonic seizures was significantly reduced from baseline at 6 and 12 months (P = 0.001), both in patients with generalized epilepsies and in patients with focal epilepsies. Significant seizure frequency reduction from baseline was observed at 12 months (P = 0.01) for tonic/atonic seizures and at 6 months (P = 0.001) for focal seizures. Side effects were reported in 21 patients (36.2%): nausea, vomiting and weight loss were most frequent. CONCLUSIONS: Rufinamide was well tolerated and was effective in reducing frequency of generalized tonic-clonic, tonic/atonic and focal seizures in both children and adults with severe refractory epilepsies, primarily LGS.

Marine fungi isolated from Chilean fjord sediments can degrade oxytetracycline.

Salmon farming is the main economic activity in the fjords area of Southern Chile. This activity requires the use of antibiotics, such as oxytetracycline, for the control and prevention of diseases, which have a negative impact on the environment. We analyzed the abilities of endemic marine fungi to biodegrade oxytetracycline, an antibiotic used extensively in fish farming. We isolated marine fungi strains from sediment samples obtained from an area of fish farming activity. The five isolated strains showed an activity on oxytetracycline and were identified as Trichoderma harzianum, Trichoderma deliquescens, Penicillium crustosum, Rhodotorula mucilaginosa, and Talaromyces atroroseus by a scanning electron microscopy and characterized by molecular techniques. Results showed significant degradation in the concentration of oxytetracycline at the first 2 days of treatment for all strains analyzed. At 21 days of treatment, the concentration of oxytetracycline was decreased 92 % by T. harzianum, 85 % by T. deliquescens, 83 % by P. crustosum, 73 % by R. mucilaginosa, and 72 % by T. atroroseus, all of which were significantly higher than the controls. Given these results, we propose that fungal strains isolated from marine sediments may be useful tools for biodegradation of antibiotics, such as oxytetracycline, in the salmon industry.

EEG extreme delta brush: An ictal pattern in patients with anti-NMDA receptor encephalitis.

INTRODUCTION: The anti-NMDA receptor (NMDAr) encephalitis-associated syndrome includes neuropsychiatric symptoms, impaired consciousness, seizures, autonomic instability, and hypoventilation. The electroencephalographic (EEG) activity throughout the course of the disease has still not been well documented. We reviewed electroclinical data of patients with NMDAr encephalitis to characterize their EEG and its clinical correlation. MATERIAL AND METHODS: We retrospectively identified 16 patients with NMDAr encephalitis from 8 Spanish medical centers, 15 of whom underwent video-EEG in the acute phase. RESULTS: In 15 patients (11 females, median age: 37.4, range: 14-87 years), seizures occurred in 9 (60%) and status epilepticus (SE) in 5 (33.3%). Magnetic resonance imaging (MRI) was abnormal in 10 (66.6%), and CSF (cerebrospinal fluid) was normal in 3 and abnormal in 12, with positive PCR (polymerase chain reaction) for Mycoplasma pneumoniae (1/15) and herpes simple virus (1/15). An ovarian teratoma was found in 1 patient and other malignancies (small cell lung carcinoma) in 1 patient. The EEG was abnormal in the acute phase in 14/15 (93.3%). Extreme delta brush (EDB) was observed in 5 (33.3%), and the presence of EDB was associated with SE in all cases. Rhythmic delta activity without EDB was observed in 5 (33.3%), while excessive beta activity was present in 4 (26.6%). Extreme delta brush can follow a pattern of well-characterized electroclinical seizures. CONCLUSIONS: Almost invariably, patients with NMDAr encephalitis had abnormal EEG. The presence of EDB, which can follow a pattern of well-characterized electroclinical seizures, in our patients was associated with seizures and SE. These findings suggest that EDB could be an evolutive pattern of an SE in NMDAr encephalitis. This article is part of a Special Issue entitled "Status Epilepticus".

Herbicides induce change in metabolic and genetic diversity of bacterial community from a cold oligotrophic lake.

Pristine cold oligotrophic lakes show unique physical and chemical characteristics with permanent fluctuation in temperature and carbon source availability. Incorporation of organic toxic matters to these ecosystems could alter the bacterial community composition. Our goal was to assess the effects of simazine (Sz) and 2,4 dichlorophenoxyacetic acid (2,4-D) upon the metabolic and genetic diversity of the bacterial community in sediment samples from a pristine cold oligotrophic lake. Sediment samples were collected in winter and summer season, and microcosms were prepared using a ration 1:10 (sediments:water). The microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated for 20 days at 10 °C. Metabolic diversity was evaluated by using the Biolog Ecoplate™ system and genetic diversity by 16S rDNA amplification followed by denaturing gradient gel electrophoresis analysis. Total bacterial counts and live/dead ratio were determined by epifluorescence microscopy. The control microcosms showed no significant differences (P > 0.05) in both metabolic and genetic diversity between summer and winter samples. On the other hand, the addition of 2,4-D or Sz to microcosms induces statistical significant differences (P < 0.05) in metabolic and genetic diversity showing the prevalence of Actinobacteria group which are usually not detected in the sediments of these non-contaminated lacustrine systems. The obtained results suggest that contaminations of cold pristine lakes with organic toxic compounds of anthropic origin alter their homeostasis by inhibiting specific susceptible bacterial groups. The concomitant increase of usually low representative bacterial groups modifies the bacterial composition commonly found in this pristine lake.

[Persistent tropical diseases among migrants]. / Maladies tropicates persistantes chez les migrants.

Several infectious diseases may remain a- or pauci-symptomatic for many years before causing major clinical manifestations. Migrants are particularly vulnerable to several persistent infectious diseases due to exposure in their country of origin and their specific living conditions. This article emphasizes neglected parasitic diseases among migrants, such as schistosomiasis, strongyloidiasis and Chagas disease. In the case of co-infection with HIV, hepatitis B and C, some of these persistent parasitosis may induce more significant morbidity. These aspects are particularly important to know as these diseases, both viral and parasitic, are particularly common among migrants.

Effect of Poly(ethylene glycol) Configuration on Microbubble Pharmacokinetics.

Microbubbles (MBs) hold substantial promise for medical imaging and therapy; nonetheless, knowledge gaps persist between composition, structure, and in vivo performance, especially with respect to pharmacokinetics. Of particular interest is the role of the poly(ethylene glycol) (PEG) layer, which is thought to shield the MB against opsonization and rapid clearance but is also known to cause an antibody response upon multiple injections. The goal of this study was, therefore, to elucidate the role of the PEG layer in circulation persistence of MBs in the naïve animal (prior to an adaptive immune response). Here, we directly observe the number and size of individual MBs obtained from blood samples, unifying size and concentration into the microbubble volume dose (MVD) parameter. This approach enables direct evaluation of the pharmacokinetics of intact MBs, comprising both the lipid shell and gaseous core, rather than separately assessing the lipid or gas components. We examined the in vivo circulation persistence of 3 µm diameter phospholipid-coated MBs with three different mPEG2000 content: 2 mol % (mushroom), 5 mol % (intermediate), and 10 mol % (brush). MB size and concentration in the blood were evaluated by a hemocytometer analysis over 30 min following intravenous injections of 20 and 40 µL/kg MVD in Sprague-Dawley rats. Interestingly, pharmacokinetic analysis demonstrated that increasing PEG concentration on the MB surface resulted in faster clearance. This was evidenced by a 1.6-fold reduction in half-life and area under the curve (AUC) (p < 0.05) in the central compartment. Conversely, the AUC in the peripheral compartment increased with PEG density, suggesting enhanced MB trapping by the mononuclear phagocyte system. This was supported by an in vitro assay, which showed a significant rise in complement C3a activation with a higher PEG content. In conclusion, a minimal PEG concentration on the MB shell (mushroom configuration) was found to prolong circulation and mitigate immunogenicity.

Targeted Microbubbles for Drug, Gene, and Cell Delivery in Therapy and Immunotherapy.

Microbubbles are 1-10 µm diameter gas-filled acoustically-active particles, typically stabilized by a phospholipid monolayer shell. Microbubbles can be engineered through bioconjugation of a ligand, drug and/or cell. Since their inception a few decades ago, several targeted microbubble (tMB) formulations have been developed as ultrasound imaging probes and ultrasound-responsive carriers to promote the local delivery and uptake of a wide variety of drugs, genes, and cells in different therapeutic applications. The aim of this review is to summarize the state-of-the-art of current tMB formulations and their ultrasound-targeted delivery applications. We provide an overview of different carriers used to increase drug loading capacity and different targeting strategies that can be used to enhance local delivery, potentiate therapeutic efficacy, and minimize side effects. Additionally, future directions are proposed to improve the tMB performance in diagnostic and therapeutic applications.

Epilepsy and pregnancy. Factors associated with epileptic seizures during pregnancy.

INTRODUCTION: The management of epilepsy during pregnancy requires optimal seizure control, avoiding the potential teratogenic effects of antiepileptic drugs. OBJECTIVES: This study aims to describe the clinical characteristics and perinatal outcomes of pregnant patients with epilepsy; to analyse the factors associated with seizures during pregnancy; to describe the most commonly used antiseizure drugs in these patients; and to analyse changes in treatment regimens in 2 periods, 2000-2010 and 2011-2018. METHODS: We conducted a prospective observational study of patients with epilepsy who reported their pregnancy between 2000 and 2018. Patients were evaluated in the first and second trimesters of pregnancy, after delivery, and at one year. Data were collected on demographic variables, epilepsy, and perinatal and obstetric variables. RESULTS: A total of 101 pregnancies were included. Patients' mean age was 32.6 years; 55.4% had focal epilepsy, 38.6% had generalised epilepsy, and 5.9% had undetermined epilepsy. We recorded 90 live births, 9 miscarriages, and 5 cases of congenital malformations, 4 of which were born to women who received valproate monotherapy. Forty patients (39.6%) presented seizures, with 16 (40%) presenting generalised tonic-clonic seizures. The variables associated with seizures during pregnancy were poor seizure control in the year prior to pregnancy (66.7% vs 15.1%; P < .001), treatment with 2 or more antiseizure drugs (30% vs 14.8%; P < .001), and untreated epilepsy (25% vs 0%; P < .001). Antiseizure medications most widely used in monotherapy were lamotrigine (n = 19; 27.1%), valproate (n = 17; 24.2%), and levetiracetam (n = 12; 17.1%). In the most recent period (2011-2018), we observed a greater proportion of patients receiving monotherapy (81.5%, vs 55.3%), as well as a decrease in the use of carbamazepine (2.3%, vs 23.1%) and valproate (20.5%, vs 30.8%); and a marked increase in the use of levetiracetam (27.3%, vs 0%). CONCLUSIONS: The factors associated with the presence of seizures during pregnancy were previous poor seizure control, treatment with 2 or more antiseizure drugs, and lack of treatment during pregnancy. The most commonly used drugs were lamotrigine, valproate, and levetiracetam, with an increase in levetiracetam use and a decrease in valproate use being observed in the later period (2011-2018).

Monodispersity Increases Adhesion Efficiency and Specificity for Ultrasound-Targeted Microbubbles.

Ultrasound molecular imaging with targeted microbubbles (MBs) can be used to noninvasively diagnose, monitor, and study the progression of different endothelial-associated diseases. Acoustic radiation force (Frad) can initiate and enhance MB adhesion at the target site. The goal of this study was to elucidate the effects of various MB parameters on Frad targeting. Monodisperse or polydisperse MBs with the immune-stealth cloaked (buried)-ligand architecture were conjugated with targeting RGD or nonspecific isotype control RAD peptides and then pumped through an αvß3 integrin-coated microvessel phantom at a wall shear stress of 3.5 dyn/cm2. Targeting was assessed by measuring MB attachment for varying Frad time and frequency, as well as MB concentration and size distribution. We first confirmed that primary Frad is necessary to target the cloaked-ligand MBs. MB targeting increased monotonically with αvß3 integrin density and Frad time. MB attachment and, to a lesser extent specificity, also increased when driven by Frad near resonance. MB targeting increased with MB concentration, although a shift in behavior was observed with increasing MB-MB interactions and aggregations forming from secondary Frad effects as MB concentration was increased. These secondary Frad effects reduced targeting specificity. Finally, after having validated our approach by testing different parameters with the appropriate controls, we then determined the effects of monodispersity on adhesion efficiency and specific targeting. We observed that both MB targeting efficiency and specificity were greatly enhanced for monodisperse vs polydisperse MBs. Analysis of videomicroscopy images indicated that secondary Frad effects may have disproportionally inhibited targeting of polydisperse MBs. In conclusion, our in vitro results indicate that monodisperse MBs driven near resonance and at a low concentration (∼106 MB/mL) can be used to maximize the adhesion efficiency (up to 88%) and specificity of RGD-MB targeting.

Effect of Anesthetic Carrier Gas on In Vivo Circulation Times of Intravenously Administered Phospholipid Oxygen Microbubbles in Rats.

OBJECTIVE: For the treatment of tumor hypoxia, microbubbles comprising oxygen as a majority component of the gas core with a stabilizing shell may be used to deliver and release oxygen locally at the tumor site through ultrasound destruction. Previous work has revealed differences in circulation half-life in vivo for perfluorocarbon-filled microbubbles, typically used as ultrasound imaging contrast agents, as a function of anesthetic carrier gas. These differences in circulation time in vivo were likely due to gas diffusion as a function of anesthetic carrier gas, among other variables. This work has motivated studies to evaluate the effect of anesthetic carrier gas on oxygen microbubble circulation dynamics. METHODS: Circulation time for oxygen microbubbles was derived from ultrasound image intensity obtained during longitudinal kidney imaging. Studies were constructed for rats anesthetized on inhaled isoflurane with either pure oxygen or medical air as the anesthetic carrier gas. RESULTS: Results indicated that oxygen microbubbles were highly visible via contrast-specific imaging. Marked signal enhancement and duration differences were observed between animals breathing air and oxygen. Perhaps counterintuitively, oxygen microbubbles disappeared from circulation significantly faster when the animals were breathing pure oxygen compared with medical air. This may be explained by nitrogen counterdiffusion from blood into the bubble, effectively changing the gas composition of the core, as has been observed in perfluorocarbon core microbubbles. CONCLUSION: Our findings suggest that the apparent longevity and persistence of oxygen microbubbles in circulation may not be reflective of oxygen delivery when the animal is anesthetized breathing air.

Actinotrichia collagens and their role in fin formation.

The skeleton of zebrafish fins consists of lepidotrichia and actinotrichia. Actinotrichia are fibrils located at the tip of each lepidotrichia and play a morphogenetic role in fin formation. Actinotrichia are formed by collagens associated with non-collagen components. The non-collagen components of actinotrichia (actinodins) have been shown to play a critical role in fin to limb transition. The present study has focused on the collagens that form actinotrichia and their role in fin formation. We have found actinotrichia are formed by Collagen I plus a novel form of Collagen II, encoded by the col2a1b gene. This second copy of the collagen II gene is only found in fishes and is the only Collagen type II expressed in fins. Both col1a1a and col2a1b were found in actinotrichia forming cells. Significantly, they also expressed the lysyl hydroxylase 1 (lh1) gene, which encodes an enzyme involved in the post-translational processing of collagens. Morpholino knockdown in zebrafish embryos demonstrated that the two collagens and lh1 are essential for actinotrichia and fin fold morphogenesis. The col1a1 dominant mutant chihuahua showed aberrant phenotypes in both actinotrichia and lepidotrichia during fin development and regeneration. These pieces of evidences support that actinotrichia are composed of Collagens I and II, which are post-translationally processed by Lh1, and that the correct expression and assembling of these collagens is essential for fin formation. The unique collagen composition of actinotrichia may play a role in fin skeleton morphogenesis.

Modelling tuberculosis trends in the USA.

We present a mathematical transmission model of tuberculosis in the USA. The model is calibrated to recent trends of declining incidence in the US-born and foreign-born populations and is used in assessing relative impacts of treatment of latently infected individuals on elimination time, where elimination is defined as annual incidence <1 case/million. Provided current control efforts are maintained, elimination in the US-born population can be achieved before the end of this century. However, elimination in the foreign-born population is unlikely in this timeframe even with higher rates of targeted testing and treatment of residents of and immigrants to the USA with latent tuberculosis infection. Cutting transmission of disease as an interim step would shorten the time to elimination in the US-born population but foreign-born rates would remain above the elimination target.

Use of ultrasound in the reproductive management of dairy cattle.

The significant decrease in fertility observed in dairy cattle during the last few decades and increasing requirements by the farmers have made a regular control of reproduction indispensable to urgently identify and solve potential problems affecting reproductive efficiency. Traditionally, the main diagnostic methods used for reproductive control in cattle included rectal palpation, inspection of vaginal discharge and vaginoscopy. Since the 1990 s, the use of ultrasound (US) has become a common diagnostic method as a result of the new advances made in the development of US scans: smaller size, high level of autonomy, high image quality and accessible prices. Ultrasound improves accuracy in the diagnoses of stages of the oestrous cycle, ovarian and uterine pathologies, and pregnancy diagnosis. In addition, it facilitates the diagnosis of alterations during pregnancy (embryo mortality, foetal malformations, etc.) and helps determining foetal sex from day 55 of pregnancy.

Insomnia and poor sleep quality are associated with poor seizure control in patients with epilepsy. / El insomnio y la pobre calidad de sueño se asocian a un mal control de crisis en pacientes con epilepsia.

OBJECTIVES: This study aimed to assess the presence of sleep disorders in patients with epilepsy and to analyse their association with seizure control. METHODS: We performed a cross-sectional study of patients with epilepsy, recruited consecutively between September 2017 and December 2018. Patients were classified as having good seizure control (no seizures in the last 4 weeks) or poor seizure control (at least one seizure in the last 4 weeks). We performed intergroup comparisons for demographic and clinical data, insomnia (Insomnia Severity Index [ISI]), excessive daytime sleepiness (Epworth Sleepiness Scale [ESS]), sleep quality (Pittsburgh Sleep Quality Index [PSQI]), depression (Beck Depression Inventory-II [BDI-II]), and quality of life (Quality of Life in Epilepsy Inventory-10 [QOLIE-10]). RESULTS: The sample included a total of 123 patients, of whom 31.7% had excessive daytime sleepiness (ESS≥10), 50.4% had insomnia (ISI≥10), and 53.6% had poor sleep quality (PSQI≥5). According to our multivariate analysis, presence of seizures was associated with unemployment (odds ratio [OR]=4.7; 95% confidence interval [CI], 1.36-19.2; P=.02), a higher number of antiepileptic drugs (OR=5.87; 95% CI, 1.81-27.1; P<.001), insomnia (OR=1.9; 95% CI, 1.1-9.3; P=.04), and poor sleep quality (OR=2.8; 95% CI, 1.9-10.32; P=.01). CONCLUSIONS: Sleep disorders are common in patients with epilepsy. Insomnia and poor sleep quality were associated with poor seizure control. These findings support the hypothesis that sleep disorders constitute a significant comorbidity of epilepsy, especially in patients with poor seizure control.

Insomnia and poor sleep quality are associated with poor seizure control in patients with epilepsy.

OBJECTIVE: This study aimed to assess the presence of sleep disorders in patients with epilepsy and to analyse their association with seizure control. METHODS: We performed a cross-sectional study of patients with epilepsy, recruited consecutively between September 2017 and December 2018. Patients were classified as having good seizure control (no seizures in the last 4 weeks) or poor seizure control (at least one seizure in the last 4 weeks). We performed intergroup comparisons for demographic and clinical data, insomnia (Insomnia Severity Index [ISI]), excessive daytime sleepiness (Epworth Sleepiness Scale [ESS]), sleep quality (Pittsburgh Sleep Quality Index [PSQI]), depression (Beck Depression Inventory-II [BDI-II]), and quality of life (Quality of Life in Epilepsy Inventory-10 [QOLIE-10]). RESULTS: The sample included a total of 123 patients, of whom 31.7% had excessive daytime sleepiness (ESS ≥ 10), 50.4% had insomnia (ISI ≥ 10), and 53.6% had poor sleep quality (PSQI ≥ 5). According to our multivariate analysis, presence of seizures was associated with unemployment (odds ratio [OR] = 4.7; 95% confidence interval [CI], 1.36-19.2; P = .02), a higher number of antiepileptic drugs (OR = 5.87; 95% CI, 1.81-27.1; P < .001), insomnia (OR = 1.9; 95% CI, 1.1-9.3; P = .04), and poor sleep quality (OR = 2.8; 95% CI, 1.9-10.32; P = .01). CONCLUSIONS: Sleep disorders are common in patients with epilepsy. Insomnia and poor sleep quality were associated with poor seizure control. These findings support the hypothesis that sleep disorders constitute a significant comorbidity of epilepsy, especially in patients with poor seizure control.

Microbubble Size and Dose Effects on Pharmacokinetics.

Optimization of contrast-enhanced imaging and focused ultrasound therapy requires a comprehensive understanding of in vivo microbubble (MB) pharmacokinetics. Prior studies have focused pharmacokinetic analysis on indirect techniques, such as ultrasound imaging of the blood pool and gas chromatography of exhaled gases. The goal of this work was to measure the MB concentration directly in blood and correlate the pharmacokinetic parameters with the MB size and dose. MB volume dose (MVD) was chosen to combine the size distribution and number into a single-dose parameter. Different MB sizes (2, 3, and 5 µm diameter) at 5-40 µL/kg MVD were intravenously injected. Blood samples were withdrawn at different times (1-10 min) and analyzed by image processing. We found that for an MVD threshold < 40 µL/kg for 2 and 3 µm and <10 µL/kg for 5 µm, MB clearance followed first-order kinetics. When matching MVD, MBs of different sizes had comparable half-lives, indicating that gas dissolution and elimination by the lungs are the primary mechanisms for elimination. Above the MVD threshold, MB clearance followed biexponential kinetics, suggesting a second elimination mechanism mediated by organ retention, possibly in the lung, liver, and spleen. In conclusion, we present the first direct MB pharmacokinetic study, demonstrate the utility of MVD as a unified dose metric, and provide insights into the mechanisms of MB clearance from circulation.

Release of vascular agonists from liposome-microbubble conjugate by ultrasound-mediated microbubble destruction: effect on vascular function.

The endothelium is a single cell layer of the vessel wall and a key regulator of blood flow in vascular beds. Local and systemic pathologies have been associated with alterations in endothelial function. However, targeting the endothelium with vasoconstrictor or vasodilator drugs is often accompanied by systemic effects. Here, we evaluated a liposome-microbubble delivery system as a vascular hydrophilic agonist carrier. Phenylephrine (Phe) or acetylcholine (Ach)-loaded liposomes were conjugated to microbubbles. The drug release was triggered by ultrasound (US), and the vascular response was assessed in rat aortic rings using an isolated organ chamber. Aortic rings incubated with Phe-liposome-microbubble conjugate, exposed to US showed a marked contractile response (0.79 ± 0.04 g) compared to empty liposomes conjugated to microbubbles, aortic rings exposed only to US, and Phe-liposome-microbubble conjugate without US exposure that elicited a minimal or no response. Expressed as %, contractile responses were 85.24 ± 4.31% and 12.62 ± 3.23% for Phe-Chol-liposome-microbubble conjugate and empty Chol-liposome-microbubble conjugate exposed to US, respectively. Addition of 1 × 10-5 M Ach to pre-contracted aortic rings decreased the contraction response from 1 to 0.21 g. The addition of Ach-liposome conjugate and exposure to US decreased the contraction response to 0.32 g. Additionally, the ED50 values for Phe and Ach released by US from liposome-microbubble conjugates were 3.6 × 10-8 M ± 2.8 × 10-9 M for Phe and 2.0 × 10-8 M ± 1.8 × 10-9 M. In conclusion, we evaluated a hybrid delivery system that consisted of loaded liposomes conjugated to microbubbles to deliver and release vascular agonists using UMMD.